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• More enzyme-conjugate added than required (increasing the concentration of HRPO), which causes the color to darken excessively. When adding the enzyme be sure to wipe the out side of pipette tip to remove excess enzyme coated on the outside. • Before adding enzyme-conjugate to conjugate diluent, be sure to mix enzyme thoroughly by pipetteing couple of times. • H2SO4 stop solution was not added early enough as directed. • Inadequate washing of the wells. • Always use distilled water for washing.
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After adding the TMB SUBS the standards darken too quickly, and/or why are the O.D. readings high?
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